Bull Semen Extender Research Articles

Cryoprotective effect of vaccenic acid supplemented in bull semen extender

Cryoprotective effect of vaccenic acid supplemented in bull semen extender

Curcumin in a tris-based semen extender improves cryosurvival of Hariana bull spermatozoa.

In this study, the cryoprotective potential of natural antioxidant curcumin in Hariana bull semen was evaluated as an additive in a tris-based extender with the assessment of motility and motion parameters of spermatozoa, membrane intactness, progesterone-receptor binding, protein carbonyl content, cervical mucus penetration, cryocapacitation-associated and apoptotic-like changes. The collected ejaculates were divided into fivegroups in the tris-based extender(control without curcumin-I, 10µM-II, 25µM-III, 50µM-IV and 75µM-V) and were cryopreserved. Groups II and III containing 10 and 25µM curcumin substantially (p<.05) improved the post-thaw sperm parameters like viability, motility, and velocity parameters; intact acrosome and membrane; lowered protein carbonyl content; DNA fragmentation and cryocapacitation-associated changes in comparison to control. It was interesting to note that early apoptotic-like changes in sperm cells were significantly (p<.05) decreased in Group II along with an increase in a higher population of sperm cells having high mitochondrial transmembrane potential. Higher progesterone-receptor binding, Vanguard distance and in vitro capacitation response were observed only in Group II (10µM) compared to other groups. In conclusion, curcumin in a semen extender manifests cryoprotective effects and may be incorporated at 10µM concentration in a Hariana bull semen extender for better post-thaw sperm quality.

The Effects of Antifreeze Protein III Supplementation on the Cryosurvival of Goat Spermatozoa During Cryopreservation.

Antifreeze protein (AFP) has been shown to have beneficial effects on frozen mammalian spermatozoa. However, rare reports have been published regarding the use of AFPs in storage of goat spermatozoa. The aim of this study was to investigate the effects of AFPIII on the quality of goat semen during cryopreservation. Ejaculates were collected from six Yunshang black goats through an artificial vagina. The collected semen was pooled, divided into five aliquots, and diluted with the commercial bull semen extender containing: no AFPIII (AFP-0, control), 1 μg/mL AFPIII (AFP-1), 10 μg/mL AFPIII (AFP-10), 50 μg/mL AFPIII (AFP-50), and 100 μg/mL AFPIII (AFP-100), respectively. Spermatozoa motility, membrane integrity, acrosome integrity, mitochondrial function, distribution of phosphatidylserine, and formation of reactive oxygen species (ROS) were measured after the freezing and thawing process. The results showed that the spermatozoa motility, membrane integrity, acrosome integrity, and mitochondrial function were significantly higher in frozen spermatozoa using the extender containing 1 μg/mL AFPIII as compared with the other groups (p < 0.05). Furthermore, the extender supplemented with 1 μg/mL of AFPIII resulted in higher viable and lower nonviable spermatozoa compared with the other treated groups (p < 0.05), after staining using Annexin V-fluoresceine isothiocyanate (Annexin V-FITC) and Propidium Iodide. No significant differences were found between these groups in relation to viable cells with lower ROS production. In conclusion, the addition of AFPIII to the freezing extender improved the post-thaw quality of goat semen. The optimal concentration used in this study was 1 μg/mL. However, excessively high concentrations of AFPIII were unable to exhibit their cryoprotective effects on goat spermatozoa. However, the presence of AFPIII cannot mitigate oxidative stress caused by the freezing and thawing process. In addition, in vitro fertilization or artificial insemination can further evaluate the effects of AFPIII on frozen-thawed goat spermatozoa.

Effects of resveratrol in bull semen extender on post-thaw sperm quality and capacity for fertilization and embryo development

Resveratrol, a potent antioxidant, can be an alternative semen extender constituent to protect spermatozoa against reactive oxygen species (ROS); however, effects on sperm quality post-thawing and sperm function is not well understood. This study, therefore, was conducted to investigate effects of resveratrol supplementation to semen extender on sperm quality post-thawing. Bull semen was cryopreserved using extenders not supplemented or supplemented with 0.05, 0.1, or 1 mM resveratrol. Supplementation of extender with resveratrol at 0.05 mM resulted in greater (P < 0.05) sperm progressive motility, average path velocity, straight linear velocity, linearity and straightness when compared with no or 1 mM supplementations. Furthermore, effects of 0.05 mM resveratrol supplementations on plasma membrane and acrosome integrity and sperm fertilization capacity using in vitro procedures were investigated. Supplementation of semen extender with resveratrol resulted in a greater (P < 0.05) proportion of frozen-thawed spermatozoa with an intact acrosome and plasma membrane. Results from in vitro fertilization studies indicated there were no differences (P> 0.05) when there was no supplementation or supplementation with 0.05 mM resveratrol on embryo development to the cleavage and blastocyst stages. In conclusion, addition of resveratrol to bull semen extender resulted in greater sperm quality post-thawing in a dose-dependent manner, with values for variables related to sperm quality being greater when there was resveratrol supplementation at the 0.05 mM concentration. Proportion of embryo developing to the cleavage and blastocyst stages after in vitro fertilization was not affected by resveratrol supplementation to semen extenders.

Cholesterol-cyclodextrin complex as a replacement for egg yolk in bull semen extender: sperm characteristics post-thawing and in vivo fertility

Egg yolk, a major semen extender constituent, lacks a defined composition, therefore, there are biosecurity concerns with use of egg yolk. Cryopreservation of bull semen without inclusion of animal protein in the semen extender, therefore, is an important consideration. Cholesterol may be delivered and incorporated into the sperm plasma membrane by cyclodextrins to protect sperm during cryopreservation. The aim of this study was to determine suitability of a cholesterol-cyclodextrin semen extender, without inclusion of egg yolk, for cryopreservation of bull semen. Bull semen was collected and cryopreserved in either egg yolk or with inclusions of three different concentrations of cholesterol-cyclodextrin complex (0.5, 1 or 2 mg/mL semen) in Tris-glycerol (TG) extender. Sperm motion characteristics examined using the computer-assisted sperm analysis, and plasma membrane and acrosome integrity examined using flow cytometry, were similar for all extenders. The inclusion of the greatest concentration of cholesterol-cyclodextrin complex (2 mg/mL semen) followed by dilution in TG extender resulted in lesser pregnancy rates (P < 0.05). There was a pregnancy rate of as great as 56 % when sperm cryopreserved in 0.5 mg/mL cholesterol-cyclodextrin Tris-glycerol extender were used for artificial insemination following imposing of a hormonal treatment regimen for synchrony of timing of ovarian functions among cows for conducting fixed-time artificial insemination (FTAI). Results indicate cholesterol-cyclodextrin Tris-glycerol extender, with a chemically defined composition and without inclusion of egg yolk, may be used to cryopreserve bull sperm with there being acceptable pregnancy rates when this semen is used for FTAI.

Effect of Different Concentrations of Tris Royal Jelly-Enriched Extender on Cooled and Post-Thawed Bull Semen

Preservation of semen either by cooling or freezing results in structural and functional damage of spermatozoa. Exogenous antioxidants are added to semen extender to counteract this damage and enhance its preservability. This study was designed to evaluate effect of adding various concentrations of Royal jelly to tris-extender on bull semen characteristics after cooling and post-freezing. Methods: Pooled bull semen were extended with tris-citrate-fructose egg yolk diluent (control: 0% Royal jelly) and various concentrations of Royal jelly (RJ: 0.05%, RJ2: 0.1%, RJ3: 0.2%, RJ4: 0.3%). Extended semen was subjected slow cooling for 4 hrs, followed by freezing. Cooled and post-thawed semen was assessed for percentage of motility, alive, abnormality, membrane integrity, and acrosome morphology. Additionally, post-thawed semen was used for artificial insemination to assess its field fertility. Results: In cooled semen, sperm motility percentage in the four concentrations of royal jelly was comparable to the control. Percentage of alive sperm was superior in the first concentration (RJ1). The second, third and the fourth concentrations (RJ2, RJ3 and RJ4) gave the best HOST percentage relative to the control. Acrosome integrity was the highest in the second concentration (RJ2), while the lowest was at the fourth concentration (RJ4) as compared to the control. Motility and percentage of alive spermatozoa in post-thawed semen was superior in (RJ1, RJ2) concentrations of royal jelly relative to the control. Also, sperm abnormalities were significantly reduced in (RJ1, RJ2). The HOST percentage was significantly higher in the fourth concentration (RJ4). Acrosome integrity was significantly higher in all concentrations and the best was in (RJ1, RJ2) compared to the control and other concentrations. Conception rate was the best in RJ1and RJ2. Conclusions: Addition of royal jelly to bull semen extender improved its quality during cooling and post-thawing.

Antimicrobial activity of silver-carbon nanoparticles on the bacterial flora of bull semen

The spermicidal effects of silver nanoparticles (AgNPs) hinder its application in the field of artificial insemination. In this study, silver-carbon NPs (Ag@C NPs) was synthesized and applied as an alternative antibiotic agent for bull semen extender. Ag@C NPs were characterized using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), atomic absorption flame spectroscopy, transmission electron microscope (TEM), and high-resolution TEM (HR-TEM). Data analysis revealed the successful synthesis of Ag@C NPs with a particle size of 1–5 nm (average particle size of 2.5 nm) embedded into carbon. The antimicrobial activity of Ag@C NPs was tested against bacteriospermia of fresh semen collected from five fertile bulls (three ejaculates/bull). Escherichia coli (E. Coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa) were isolated from fresh semen samples and identified by culture, staining, and conventional biochemical tests. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Ag@C NPs against bacteriospermia was determined at 5 and 37 °C. Ag@C NPs showed efficient antimicrobial activity (MIC: 3.125–12.5 μg/mL) against the tested strains and strong bactericidal effect on S. aureus, and P. aeruginosa (MBC: 3.125 μg/mL), with no detrimental effect (P ˃ 0.05) on the percentage of sperm motility (70.71 ± 4.82; 74.65 ± 4.46), plasma membrane integrity (68.39 ± 4.31; 72.38 ± 4.91), acrosome integrity (88.40 ± 13.21; 86.77 ± 14.23), and normal sperm morphology (86.85 ± 7.43; 87.82 ± 8.15) at concentrations of 15 and 30 μg/mL, respectively, after a cold storage of 48 h. However, Ag@C NPs showed a detrimental effect on sperm parameters in a dose dependent manner at concentrations ≥60 μg/mL. Ag@C NPs showed no adverse effect on the sperm’s ultrastructure with limited sperm internalization at MIC. In conclusion, Ag@C NPs could be used as an alternative antibiotic agent for bull semen extender without a significant cytotoxic effect on the sperm during cold storage. However, further investigations for their effects on embryo production and female genitalia are still required.

Effects of Leucosporidium-derived ice-binding protein (LeIBP) on bull semen cryopreservation.

We examined the effect of ice‐binding protein derived from Leucosporidium (LeIBP) on the cryopreservation of bull semen and compared it with that derived from previously reported Antifreeze Protein III (AFPIII). Six concentrations of LeIBP (10–1 ~ 104 μg/ml) and AFPIII (10–1 ~ 104 μg/ml) were added to the bull semen extender, respectively. Sperm kinematic parameters were measured to examine sperm toxicity and cryopreserved sperm quality. Measures of antioxidant activity such as superoxide dismutase (SOD), reduced glutathione/oxidative glutathione (GSH/GSSG), and total antioxidant capacity (TAC) were analysed to identify the effect of LeIBP on sperm quality. In addition, sperm viability was analysed using a flow cytometer and fluorescence microscope by SYBR14/PI staining. The results showed that the LeIBP groups (0.1, 1 and 10 μg/ml) were less toxic, and the quality of the sperm were dramatically improved in the extenders containing 0.1 μg/ml LeIBP among concentrations of LeIBP and AFPIII. The SOD activity of LeIBP was greater than that of AFPIII and control. In addition, sperm viability was enhanced in the LeIBP‐treated group. In summary, LeIBP is a useful cryoprotective adjuvant for bull sperm cryopreservation, and the most efficient concentration of LeIBP is 0.1 μg/ml.

Effect of vitamin E addition to frozen Simmental bull semen extender on post-thawing quality

The purpose of this research was to evaluate the post-thawing quality (sperm motility, mortality and abnormality) of frozen-thawed Simmental bull semen with the addition of vitamin E in the extender. The material used for research were semen from two selected Simmental bulls. Vitamin E as treatment in addition to the extender consisted of T0 (no addition of vitamin E), T1 (0,134 gram/100 ml extender), T2 (0,268 gram/100 ml extender) and T3 (0,402 gram/100 ml extender). Post-thawing evaluation conducted 24 hours after the freezing process and observed after the thawing process in 37°C water bath for 30 seconds. Parameters observed in this research were the post-thawing quality of frozen-thawed Simmental bull semen based on motility, mortality and abnormality percentage. Sperm motility evaluated using a microscope with 100x and 400x magnifying, sperm mortality observed using 400x magnifying and counted based on 0,2% eosin-negrosin staining, sperm abnormality observed using 400x magnifying and counted based on the number of morphologically abnormal sperm cells. Semen post-thawing motility was not significantly affected by vitamin E addition of T1, T2 and T3 (P&lt;0,05). T1 and T2 were able to significantly lower the mortality percentage compared to T0 and T3 (P&lt;0,05). T1 and T2 were also very significantly affecting the decreased of abnormality percentage compared to T0 and T3 (P&lt;0,01). T1 generally gave the best result on the improvement of post-thawing motility and decreased the percentage of sperm mortality and abnormality.

26 Baobab oil supplemented extender preserves post-thaw bull sperm quality parameters

The processes of semen cryopreservation and thawing affect sperm membrane integrity and motility and increases morphological defects as well as DNA damage. The most influential cause of this is oxidative stress. When endogenous antioxidant capacity of seminal plasma is reduced during the freeze-thawing process, plant extracts exhibiting strong antioxidant activity can be used as supplements for compensation. Baobab oil has gained interest because it is rich in powerful antioxidants, which could protect sperm cells from oxidative damage during cryopreservation. Our study aimed to assess the effects of baobab oil on post-thaw sperm quality parameters in an egg-yolk-based extender. Thirty semen ejaculates were collected from 15 Nguni bulls using an electro ejaculator. Semen samples were randomly allocated to control (no baobab oil), 20μL (1%), 50μL (2.5%), and 100μL (5%) baobab oil per millilitre extender. Following dilution, semen samples were loaded into 0.25-mL semen straws, equilibrated for 4h at 5°C, and transferred into a controlled rate programmable freezer. The frozen semen straws were stored in a liquid nitrogen tank (−196°C) until thawing. Semen straws were thawed (37°C/60 minutes) after 1 week of cryopreservation and analysed for (1) sperm motility using a computer-aided sperm analyser, (2) morphological defects and viability using eosin-nigrosin stain, (3) membrane integrity by hypo-osmotic swelling test, and (4) DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. Data was analysed using analysis of variance. Treatment means were compared in relation to the control group by Dunnett's test. We found that supplementing semen extender with baobab oil at 1% significantly (P&amp;lt;0.05) preserved sperm DNA integrity (88.3±3.7) and membrane integrity (74.0±4.2) when compared with the control group (71.7±3.7 and 55.8±4.4, respectively). Baobab oil supplementation either at 1% (5.9±0.5), 2.5% (7.2±0.5), or 5% (6.0±0.5) significantly reduced sperm morphological defects compared with control (9.5±0.5). Total motility (1% (72.7%), 2.5% (72.7%), 5% (71.9%), control (59.3%)) and viability (1% (79.1%), 2.5% (79.8%), 5% (77.8%), control (67.6%)) were also improved by supplementation; however, the difference was not significant. In conclusion, it was demonstrated that supplementing bull semen extender with 1% baobab oil protects sperm from morphological defects, maintains membrane integrity, as well as preserves sperm DNA. All the baobab oil supplementation levels preserved post-thaw bull-sperm quality parameters.

Improving the quality of cryopreserved goat semen with a commercial bull extender supplemented with resveratrol

The purpose of the current study was to evaluate the effects of resveratrol (RSV) on the quality of frozen-thawed goat sperm. Semen samples from four bucks were divided into five aliquots and diluted with a commercial bull semen extender containing: no antioxidant (RSV-0, control), 10 μM RSV (RSV-10), 50 μM RSV (RSV-50), 100 μM RSV (RSV-100) and 250 μM RSV (RSV-250). After thawing, sperm motility, abnormal morphology, membrane and acrosome integrity, mitochondrial activity, phosphatidylserine (PS) distribution, and oxidative stress were evaluated. The results indicated that in comparison with the control, when the concentration of RSV was 10 or 50 μM, the total motility, progressive motility, membrane and acrosome integrity, and mitochondrial activity of post-thaw spermatozoa was greater (P < 0.05). Additionally, the use of extenders containing RSV-10 or RSV-50 resulted in a greater percentage of viable spermatozoa as compared to the other groups (P < 0.05). Importantly, there were more viable spermatozoa (49.61 ± 0.61%) and less non-viable spermatozoa (49.16 ± 1.01%) in the RSV-50 group compared to the other extenders (P < 0.05). Furthermore, the use of the extenders containing RSV-10 and -50 resulted in a reduction in ROS production in frozen-thawed spermatozoa as compared to the control (P < 0.05). There, however, was no difference among extenders for abnormal morphology and PS distribution. In conclusion, supplementation with RSV, at a concentration of 10 or 50 μM in the semen extender, can improve the post-thaw goat sperm quality, which may occur as a consequence of inhibition of ROS generation.

Effect of reduced glutathione supplementation in semen extender on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa

To provide new insights into the mechanisms through which reduced glutathione (GSH) is able to protect spermatozoa, we tested the hypothesis that cryocapacitation and apoptosis like changes can contribute to the negative effect of freezing and thawing on bull spermatozoa, and that GSH prevent this damage. Having known protective effects of GSH in terms of a potent antioxidant, we evaluated capacitation, tyrosine phosphorylation and apoptosis like changes in bull spermatozoa after freezing and thawing in egg yolk tris glycerol extender containing (0.5m M-GSH-T1 & 1mM GSH-T2) and without GSH serving as the control (C). Forty ejaculates were collected from four Hariana bulls and were pooled due to non significant variations among the bull ejaculates for the evaluation of sperm attributes. Capacitation like changes, tyrosine phosphorylation, localization of tyrosine phosphorylated proteins, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA fragmentation after final dilution, 4h of equilibration at 4°C and 24h after freezing and thawing were evaluated. GSH supplementation at 0.5mM showed significant reduction in B- and AR- pattern spermatozoa during all stages of semen freezing and thawing. Immunoblot revealed six proteins which were tyrosine phosphorylated and protein of 30 and 75kDa (p30, p75) were the major tyrosine phosphorylted proteins. On further analysis, the p30 showed differential variation in intensity in all the three groups after freezing and thawing. Positive immune reactivity for tyrosine phosphorylated proteins was found in neck, middle piece and post-acrosomal regions of spermatozoa. Addition of 0.5mM GSH decreased percentage of spermatozoa showing fragmented DNA and increased the percentage of spermatozoa having high transmembrane mitochondrial potential (P<0.05). This study demonstrates that GSH favours survival of bull spermatozoa by interfering with apoptotic and cryocapacitation pathways, and thereby protects the spermatozoa from deleterious effects of cryopreservation. The findings of the study indicated that GSH at 0.5mM can be effectively used as an additive in bull semen extender for freezing and thawing.

Substitution of egg yolk with different concentrations of soybean lecithin in tris-based extender during bulls' semen preservability

ObjectiveTo investigate the effect of various concentrations of soybean lecithin as an alternative for egg yolk in bull semen extender on post-chilling and post-thaw sperm quality characteristics. MethodsSemen ejaculates were collected from three mature bulls' once/week for 5 weeks. After initial evaluation the approved ejaculates were pooled and extended gradually 1:7 with tris-citrate-fructose egg yolk extender (control) and tris-citrate-fructose (TCF)+different concentration of soybean lecithin (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5% and 4.0%) to ensure 60 million motile spermatozoa/mL, and then proceeded an adopted international cryopreservation protocol. Frozen straws were thawed at 37 °C for 30 s. The parameters studied were sperm motility, viability, abnormality, membrane integrity and normal intact acrosome percentages in chilled and frozen-thawed semen. ResultsThe substitution of egg yolk into TCF with 1% soya lecithin significantly (P < 0.0001) ameliorated the maintenance of semen characters motility (86.67% ± 0.80%), life (87.60% ± 0.88%), and membrane integrity (82.47% ± 0.94%), meanwhile it had significantly (P < 0.0001) reduced the abnormality (13.20% ± 0.60%) of spermatozoa to its least value compared to some other concentrations in use. Moreover, the addition of 1.5% of soya lecithin to TCF had maintained the semen characteristics compared to the control tris-citrate-fructose egg yolk. The significantly higher mean values of post-thawing sperm motility % were observed in 1% soybean lecithin (56.00% ± 1.00%) as compared to the control (41.00% ± 1.00%). Conclusion1–1.5% soya lecithin can effectively alternate egg yolk as cryoprotective additives for cryopreservation extender, without any detrimental effects on post-chilling and post-thaw semen quality in cattle bull.

Effects of pomegranate juice in Tris-based extender on cattle semen quality after chilling and cryopreservation

ObjectiveTo study the effect of adding different concentrations of the pomegranate juice (PJ) to the cattle bull semen extender on post-thawing semen quality. MethodsSemen was collected from five cattle-bulls at weekly intervals for 5 weeks at the Semen Freezing Center, General Organization for Vet. Services, Ministry of Agriculture. Semen samples were diluted in Tris-citric acid-egg yolk-fructose extender and divided into six aliquots, the 1st served as control while PJ was supplemented at 10%, 20%, 30%, 40% and 50% in the aliquot 2, 3, 4, 5 and 6 respectively. Diluted semen samples were subjected to cooling and cryopreservation and stored in liquid nitrogen (LN2). Sperm motility in chilled semen (over 10 d) and post-thawing sperm parameters, including individual motility, alive sperm, membrane integrity, and total sperm abnormality were assessed. ResultsObtained results clearly demonstrated that the addition of 10% PJ in the chilled extended cattle semen proved to be beneficial for maintaining sperm motility percentage. On the other hand, the addition of 40% and 50% PJ failed to preserve motility all over the 10 d. Also, supplementation of extender with 10–20% PJ significantly increases the post-thaw motility and viability as compared with control group. ConclusionsSupplementation of bull semen extender with 10% and 20% PJ provides good chilling and improved frozen-thawed semen quality.

Comparative Quality Assessment of Buffalo (Bubalus bubalis) Semen Chilled (5°C) in Egg Yolk‐ and Soya Milk–Based Extenders

Egg yolk-Tris is most commonly used semen extender; however, its use involves hygienic risk, interference with fertility and poor microscopic examination. Therefore, replacement of egg yolk with a plant-based component with protective effects on spermatozoa would be advantageous. In present study, we observed effect of soya milk-based extenders on dilution and liquid preservation of Murrah buffalo bull semen at 5°C up to 72 h in comparison with conventional egg yolk-Tris extender (Ext.1). In experiment one, a total of 32 buffalo semen ejaculates from four animals were extended and preserved at 5°C for 72 h in soya milk-based extender (Ext.2) with different percentages (10%, 15%, 20%, 25% and 30%) of soya milk for optimization of soya milk concentration. Semen quality was assessed for individual motility, viability, membrane integrity and acrosome integrity at 0, 24, 48 and 72 h of liquid preservation. The results of experiment one indicated that 25% soya milk is an optimum concentration for buffalo bull semen extender preparation. A modified method was used to prepare another soya milk-based extender (Ext.3). In the second experiment, two soya extenders (Ext.2 and 3) with optimized concentration (25%) of soya milk were comparatively assessed with egg yolk-Tris extender (Ext.1) for semen quality parameters at 0, 24, 48 and 72 h of liquid preservation. The individual sperm motility at 0 and 24 h following dilution were found non-significant among extenders. However, after 48 h of dilution, individual motility in Ext.3 was observed significantly (p < 0.05) higher than Ext.1. After 24, 48 and 72 h of dilution sperm membrane integrity in Ext.3 was found significantly (p < 0.05) higher than Ext.1. Overall, comparative evaluation of sperm parameters obtained revealed that Ext.3 containing 25% soya milk can be used as a substitute of egg yolk-based extender for buffalo semen liquid preservation.

Effects of curcumin and dithioerythritol on frozen-thawed bovine semen

The aim of this study was to determine the effects of curcumin and dithioerythritol added into bull semen extender on sperm parameters, lipid peroxidation, total glutathione and antioxidant potential levels of bull spermatozoa following the freeze/thawing process. Twenty-seven ejaculates obtained from three bulls were included in the study. Each ejaculate that was splitted into five equal groups and diluted in a Tris-based extender containing curcumin (0.5 and 2 mM), dithioerythritol (0.5 and 2 mM) and no additive (control) was cooled to 5 °C and frozen in 0.25-ml French straws. The extender supplemented with 0.5 mMdose of curcumin led to lower percentage of total abnormality (20.40 ± 2.36%) when compared to the control (30.60 ± 1.47%, P < 0.05). Curcumin and dithioerythritol at 0.5 mM provided a greater protective effect in the membrane functional integrity (54.40 ± 2.09% and 50.00 ± 2.68%), in comparison with control (37.20 ± 1.77%, P < 0.001). Supplementation with antioxidants did not significantly affect the lipid peroxidation and antioxidant potential levels, while the maintenance of total glutathione levels in curcumin 0.5 mM was demonstrated to be higher than that of control, following the freeze/thawing (P < 0.05). Supplementation with these antioxidants prior to the cryopreservation process may be recommended to facilitate the enhancement of sperm cryopreservation techniques.

95 EFFICACY OF FIVE DIFFERENT SEMEN EXTENDERS FOR THE CRYOPRESERVATION OF BULL SEMEN

Replacement of animal-origin components in extenders used for bull semen freezing is of high importance for individuals involved in cattle breeding. The experiment was designed to compare efficacy of 5 different semen extenders in cryopreservation of bull semen: sodium citrate-based extender containing egg yolk (CT), commercially available Bioxcell� (IMV Technologies, L'Aigle, France), and 3 custom-made homogenized plant lipidsbased, egg yolk-free extenders (Y-1, Y-2, and Lipo) . The objective was to determine whether homogenization procedures of lipids improve the quality of the extender. Lipid homogenates of custom-made extenders were prepared in Tris buffer using a high pressure homogenizer (Nira Saovi, Parma, Italy). Ten (Y-1) or 5 (Y-2) homogenization cycles were applied and then 8% glycerol was added. Lipid liposomes were produced by simultanous high pressure homogenization of lipids and glycerol supplementation (Lipo). Semen was collected from young bulls of 3 different breeds (Simmental, Polish Red, and Holstein; 1 ejaculate/bull). Each ejaculate with at least 70% motility was split into 5 parts and processed further by a standard freezing protocol: semen was diluted at 35�C with each of the 5 extenders to a concentration of 100 � 106 spermatozoa per mL, cooled to 4�C over 5 h, aspirated into 0.25-mL plastic straws, frozen in liquid nitrogen vapor to –140�C, and then plunged into LN2. Straws were thawed in a water bath at 37�C for 20 s. Sperm motility was estimated microscopically immediately after thawing and after 5 h of storage at 22�C. Immediately after thawing, flow cytometry and SYBR-14/PI staining were used for examination of sperm membrane integrity (live/dead assay). A total of 20 000 spermatozoa of each sample were counted. Student's t-test was used to estimate statistical differences between experimental groups. The mean sperm motility after thawing ranged from 45.6% (SD = 13.7) for CT (egg yolk extender) to 57.8% (SD = 7.1) for Lipo. A significant difference (P &lt; 0.05) was observed betweenY-1 (50.0%, SD = 9.7) and Lipo and Bioxcell (56.1%, SD = 8.6). After 5 h of storage at 22�C, the mean motility for all tested bulls ranged from 25.0% (SD = 7.1) for CT to 42.2% (SD = 7.5) for Lipo. Significant differences were observed between Lipo (P &lt; 0.01), Y-2 (P &lt; 0.05) and CT, and between Y-1 and Lipo (P &lt; 0.01). Mean percentage of 'live' spermatozoa with intact membrane after freezing/thawing was 51.85% (SD = 11.49) for Y-1, 45.72% (SD = 9.36) for Y-2, 47.57% (SD = 7.93) for Lipo, 45.47% (SD = 8.35) for Bioxcell, and 49.06 (SD = 11.59) for CT. No significant differences were observed except forY-1 and Bioxcell extenders (P &lt; 0.05). It can be concluded that both methods of lipid/glycerol homogenization can be successfully applied in the preparation of bull semen extender. In addition, extensive lipid homogenization (10 cycles) produced more transparent extender that in turn improved visualization of sperm. Custom-made plant origin lipids homogenization may provide a valuable alternative for the preparation of extenders that more closely match the membrane composition of bull sperm cells and thus contribute to development of an efficient extender free of animal-origin components for bull semen freezing.

98CONCEPTION RATES USING BRAHMAN BULL SEMEN FROZEN IN MILK BASED EXTENDER CONTAINING EGG YOLK OR SOYBEAN LIPIDS; A FIELD STUDY IN A TROPICAL ENVIRONMENT

The objective of this study was to examine the substitution of soybean-origin phospholipids for egg yolk in Brahman bull semen extender. Semen was frozen in 3 different low-fat milk (1%) based extenders containing 10mgmL−1 of fructose and supplemented with: 8% of whole egg yolk (Extender 1, control), 8% rectified egg yolk (egg yolk granules were removed by double centrifugation at 3000g for 1h at 5°C; Extender 2), and 7.3mgmL−1 of phospholipids of soybean-origin containing 10% of phosphatidyl choline (Extender 3). All 3 extenders were supplemented with 1000IU of penicillin, 1mgmL−1 streptomycin and 150μgmL−1 lincomycin. The semen was collected by means of artificial vagina from 3 Brahman bulls, and AI was performed during the dry season between December and April in a tropical forest environment. The mean temperature for the region was 26–30°C, with mean rainfall of 900–1500mm/year and the relative humidity of 60–70%. Ejaculates with at least 60% motility were diluted in 2 steps as follows: in step 1, each ejaculate was split into 3 even parts and diluted at 26°C with each of the extenders containing no glycerol, and in step 2, 14% of glycerol was added in 15-minute intervals to a final glycerol concentration of 7%. Semen was aspirated into 0.5mL plastic straws (20×106 sperm/per straw), frozen 7cm above liquid nitrogen (LN2) for 8min, and then plunged into LN2. Straws were thawed in a water bath at 37°C for 30s. Each experiment was replicated 3 times (different collection days). Sperm viability was tested within artificial insemination trials. Results are based on the pregnancy rates of crossbreed Brahman cows determined by palpation 45 Days after AI and by calving rates. Data were compared by chi-square analysis. In Experiment I, a total of 157 cows were inseminated with semen collected from 3 different bulls (A, B and C) and frozen in 3 different extenders (1, 2 and 3; 3×3 factorial design). Bull A, Extender 1, 2 and 3 (n=19, 20 and 22); Bull B, Extender 1, 2 and 3 (n=20, 20 and 20) and Bull C, Extender 1, 2 and 3 (n=22, 15 and 24), respectively. Although semen from all 3 bulls frozen in Extenders 2 and 3 fostered numerically higher pregnancy rates (from 30% for Bull B and Extender 2 to 50% for Bull C and Extender 3) than in Extender 1 (from 23.5% for Bull C to 40% for Bull B), there were no differences (P&amp;lt;0.05) between bulls with any of 3 extenders on the pregnancy rates. In Experiment II, a total of 117 cows were inseminated with semen collected from Bull B and frozen in Extender: 1 (n=37), 2 (n=48) and 3 (n=39). There were significantly higher (P&amp;lt;0.05) calving rates for cows inseminated with semen frozen in Extender 2 and 3 (41.6% and 46.1%, respectively) than in Extender 1 (24.3%). It can be concluded that rectified egg yolk may improve viability of frozen semen, and that phospholipids of soybean origin can be successfully substituted for egg yolk in Brahman bull milk based semen extender. Supported by Bioniche Inc, Belleville, Ontario, Canada.